Thanks Liz, You are absolutly right there, but have you ever noticed some folks eyes glaze over when you say that they must both be run at the same ug/mL? I would be lying if I said it never happened to me until I forced myself to work it out. Once you do though it isn't too bad. Unfortunately people don't think about this much. They want to put a slide on a machine and walk away without thinking about what they are doing. I guess I was oversimplifying the description. Thanks for pointing that out as it is actually an important aspect to the dilutions.
Amos On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala <l...@premierlab.com> wrote: > Amos > > Isotype negative controls are based upon protein concentration not > dilution. They must be the same protein concentration of the primary > antibody at the dilution you are using. Using the same dilution will > not work unless both stock solutions (primary antibody and isotype > control) are at the same protein concentration which is rarely the case. > > Liz > > Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC > Manager > Premier Laboratory, LLC > PO Box 18592 > Boulder, Colorado 80308 > office (303) 682-3949 > fax (303) 682-9060 > www.premierlab.com > > > Ship to Address: > 1567 Skyway Drive, Unit E > Longmont, Colorado 80504 > > -----Original Message----- > From: histonet-boun...@lists.utsouthwestern.edu > [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos > Brooks > Sent: Friday, May 20, 2011 2:16 PM > To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu > Subject: [Histonet] IHC pos. & neg. control question > > Hi, > This is simply a question of definitions. They are actually both > right. > If you have the patient tissue as a negative control you are not using > the > primary antibody on it but are replacing it (ideally) with an Ig with > the > same isotype AND dilution (universal negative is stupid). So if your > primary > is an IgG1 from a mouse and you use it at 1:100, your negative control > would > be a non-immune mouse serum IgG1 diluted to 1:100. Running this on > unrelated > material will not show you anything in this case. what it will show you > is > that the detection system you are using is specific to the antibody you > put > on the tissue and not to A) something else in the tissue or B) something > on > the Ig that is not the epitope itself. > Although, If you run the same primary antibody on tissue that you > expect will not react with the primary antibody, this would prove that > the > antibody reaction is specific to the antigenin question. This does raise > the > question of what to do about ubiquitous antigens (like ubiquitin) that > are > actually everywhere. Every cell has a cell cycle so they will all at one > point or another show proliferation or degeneration for example. Does > this > mean you don't run a negative control? No you need to look at it in the > perspective of expected results. > There are various ways of using negative (and positive) controls. > It > would actually be cost prohibitive to run ALL the possible positive and > negative controls for every test that we as histologists do. We need to > discuss with our pathologists what they want to have done to give them > the > confidence in our testing to support their diagnosis. But remember that > just > because another lab and another pathologist does it differently doesn't > mean > it is wrong. It's just different. > > Happy Friday Folks, > Amos > > > Message: 5 > Date: Thu, 19 May 2011 13:25:49 -0400 > From: "Jean-Martin Lapointe" <jm.lapoi...@accellab.com> > Subject: [Histonet] IHC pos. & neg. control question > To: <histonet@lists.utsouthwestern.edu> > Message-ID: > < > befd613bd39142499989f836556ddc8301272...@ace.accellab.lan> > Content-Type: text/plain; charset="iso-8859-1" > > Hi Curt, > I agree with your pathologist. The section that you use as a negative > control (without primary) in an IHC run should ideally be tissue that is > a > known positive, and of the same nature as the test sample. Therefore the > best sample is often a section of your positive control tissue. > The purpose of this negative control is to make sure that any positive > staining observed in the test sample is not due to a spurious > cross-reaction, unrelated to the primary. I don't think the issue per se > is > that you are using tissue from the same patient; rather, it is that you > are > using a sample for which the staining characteristics are not known. For > instance, if are using a lymph node section as a negative, for a stain > that > targets an epithelial marker (eg Her2) in the test breast sample, then > your > negative tissue is not appropriate, because since lymph node contains no > epithelial tissue, it will not stain no matter what. Therefore if your > test > sample shows a positive reaction in the epithelial tissue, but for some > reason that reaction is a spurious false-positive, then the lymph node > negative will not reveal that. > I realize that this is all very theoretical and hypothetical, but I > understand the pathologist wanting to be confident in the knowledge that > all > potential technical issues are eliminated before making his diagnosis. > > Jean-Martin > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet